New insights into Eragrostis curvula's sexual and apomictic reproductive development

Abstract

Apomixis, defined as asexual propagation by seeds, is considered of great importance for agriculture as it allows the fixation of desired traits and its propagation through generations. Eragrostis curvula (Schrad.) Ness, is a perennial grass that comprises a polymorphic complex including sexual and diplosporous apomictic cytotypes, where all apomicts are polyploids. In this study we present the first detailed description of female and male gametophyte development in E. curvula through confocal laser microscopy, contrasting three genotypes: the fully apomictic Tanganyika, the facultative apomictic Don Walter, and the sexual OTA-S. Moreover, we have studied the localized expression of a gene known as SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), that was found to be differentially expressed in contrasting genotypes of E. curvula. This gene had been previously linked with flower development and abiotic stresses in several species, thus, in situ hybridizations were carried out in the model plant Arabidopsis thaliana, as well as in sexual and apomictic E. curvula genotypes. Our microscopy analysis has led to the identification of specific morphological characteristics for each genotype, mainly depicting a larger ovule in the sexual genotype's reproductive development after the meiosis stage. These results reveal potentially important features, which could be used for a simple identification of genotypes. Moreover, differential expression of the gene SPL7 was detected, specifically determining an overexpression of the gene in the sexual genotype. These results demonstrated that it could be an interesting candidate to understand the mechanisms behind apomictic development.

Keywords: Eragrostis curvula; apomixis; confocal laser microscopy; ovule development; pollen development; sexual reproduction.

Figure 1

Confocal laser images of ovules of OTA, Tanganyika (T) and Don Walter (DW). Scale bars of 25µm. (A, B) MMC stage in OTA; (C, D) MMC stage in Tanganyika; (E, F) MMC stage in Don Walter. The nuclei of the MMC is indicated with an arrow in (A, C, E). (K) Tetrad stage in OTA; (L) Closer images of the four nuclei of Tetrad stage in OTA. (M) Degenerating megaspores in OTA(*); (N) Functional Megaspore stage in OTA. The bar graphs indicate the comparative lengths of the MMC stage in micrometers (µm). (G, H) Measurements of the total ovule for each genotype; (I, J) Measurements of the embryo sac for each genotype. Statistical differences between genotypes are shown with a *p<0,05.

Figure 2

Confocal laser images of ovules of OTA, Tanganyika (T) and Don Walter (DW). Scale bars of 25µm. (A, B) FG2 stage in OTA; (C, D) FG2 stage in Tanganyika; (E, F) FG2 stage in Don Walter; (K, L) FG4 stage in OTA; (M, N) FG4 stage in Tanganyika; (O, P) FG4 stage in Don Walter; (U, V) FG7 stage in OTA; (W, X) FG7 stage in Don Walter; (C2) Detail of chromosomes in antipodal cells of sexual genotype OTA. The bar graphs indicate the comparative lengths of the different stages in micrometers (µm). (G, H) Measurements of the total ovule in FG2 stage; (I, J) Measurements of the embryo sac in FG2 stage; (Q, R) Measurements of the total ovule in FG4 stage; (S, T) Measurements of the embryo sac in FG4 stage; (Y, Z) Measurements of the total ovule in FG7 stage; (A2, B2) Measurements of the embryo sac in FG7 stage. Statistical differences between genotypes are shown with a *p<0,05.

Figure 3

Maximum likelihood phylogenetic tree (bootstrap = 1000) of the SPL proteins constructed with Eragrostis curvula (shown in blue), Oryza sativa (shown in green) and Arabidopsis thaliana SPLs (shown in red). Bootstrap support values greater than 50 are shown at nodes. The transcript found to be differentially expressed in E. curvula is indicated with a red star (★).

Figure 4

Confocal laser images of pollen grains of OTA, Tanganyika (T) and Don Walter (DW) for the following stages of development: Pollen mother cell (PMC); Meiosis/Pre-meiosis; Uninucleate and Binucleate pollen. Nuclei of Binucleate stages are indicated with a red arrow (➔). Scale bars of 25µm. (A) Detail of the chromosomes in pollen grains of genotype OTA.

Figure 5

Schematic summary of the differential features observed under confocal microscopy, for each developmental stage in the ovule of the three studied genotypes of E. curvula.

Figure 6

(A, B) Images of the in situ hybridizations performed in A. thaliana, for both diploid and tetraploid backgrounds of Wild Type Col-0. (*) Detected color signal. (C) Agarose gel (1,5% m/v) obtained for the amplification of the RT-PCR of sexual (O1 and O2) and apomictic (T1 and T2) cDNA duplicates with the gene SPL7. (D) Images of the in situ hybridizations performed E. curvula ovules of the sexual diploid accession PI208214 and the apomictic genotype Tanganyika, (*) Detected color signal.